DESCRIPTION Neurons express the endothelial type of NO synthase (eNOS), where it plays an important role in memory and learning. This proposal investigates the relationship between cholinergic muscarinic receptors and the activation of endothelial nitric oxide synthase. Specifically, two central hypotheses will be tested. (1) eNOS activation is coupled to muscarinic receptors in a receptor subtype selective fashion. (2) Due to the unique association of eNOS with the cell membrane, its activation might be the result of both Ca/2+ influx and phosphoinositide hydrolysis-mediated Ca/2+ mobilization. These hypothesis will be tested using transfected cells which co-express eNOS and individual muscarinic receptor subtypes. Three species aims will be pursued: (1) Preparation of transfected CHO cell lines which stably co- express eNOS and the individual subtypes of muscarinic receptors; (2) Comparison of coupling of individual muscarinic receptor subtypes (m1-m5) to the production of nitric oxide via eNOS activation; (3) Determine dependence of muscarinic receptor activation of eNOS on Ca/2+ and the mechanisms underlying generation of the C/2+ signal. These studies will advance our understanding of the cellular mechanism responsible for producing nitric oxide via eNOS. Activation of eNOS has been shown to play a role in vasodilation in the CNS and an learning and memory. Information derived from the planned studies may help develop therapies in stroke, ischemia, hypertension, and Alzheimer's disease.